The cell division cycle is one of the most fundamental processes in biology which, in multicellular organisms, ensures the controlled generation of cells with specialized functions. Under normal growth conditions, cell proliferation is tightly regulated in response to diverse intra- and extracellular signals. The Cdc25A phosphatase plays a critical role in cell cycle progression due to its function in dephosphorylating cyclin-dependent kinases, thus serving as a rate-limiting mitotic activator. Conversely, in response to DNA damage or stalled replication, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases activate the checkpoint kinases (Chk1 and Chk2) leading to Cdc25A hyperphosphorylation (Falck et al., Nature. 2001;410:842-7; Zhao et al., Proc Natl Acad Sci USA, 2002;24:24; Sorensen et al., Cancer Cell, 2003;3:247-58). These events stimulate the ubiquitin-mediated proteolysis of Cdc25A (Falck (2001) supra; Mailand et al., Science, 2000;288:1425-9; and Molinari et al., EMBO Rep, 2000;1:71-9) and contribute to delaying cell cycle progression, thereby preventing genomic instability (Falck (2001), supra; Zhao, supra; Sorensen, supra; Mailand, supra; Molinari, supra; Bartek & Lukas, Curr Opin Cell Biol, 2001;13:738-47; and Falck et al., Nat Genet, 2002;30:290-4). Dysregulation of Cdc25A has also been associated with certain types of breast cancer (Cangi et al., J Clin Invest, 2000;106:753-61).
The proteolysis of cellular regulatory proteins such as Cdc25A is a multistep process orchestrated by the concerted action of three enzymes, all leading up to the addition of a ubiquitin peptide to the protein, and subsequent transfer of the ubiquitinylated protein to a cellular structure called the proteasome where it is proteolyzed. The enzymes responsible for recruitment of each particular type of target protein to be proteolyzed are called ubiquitin ligases. Given the diversity of target proteins, there is an equally large number of ubiquitin ligases. One type is called the SCF ubiquitin ligases. The SCF ubiquitin ligases comprise three subunits; a Skp1 protein, a Cul1 protein, and an F-box protein. For a review, see Pagano and Benmaamar, Cancer Cell 2003;4:1-6. Many F-box proteins have been identified and characterized (see, e.g., WO 00/12679 by Chiaur et al., and WO 02/055665 by Pagano et al.), which is often the SCF subunit responsible for targeting the target protein to be proteolyzed.
It has previously been shown that the ubiquitin-mediated degradation of Cdc25A at the exit of mitosis is mediated by the APC/CCdh1 (Anaphase Promoting Complex/Cyclosome) ubiquitin ligase through recognition of a specific KEN box sequence (Donzelli et al., Embo J, 2002;21:4875-84). In the same study, it was also demonstrated that a Cdc25A KEN mutant is resistant to APC-mediated ubiquitylation, yet it remains short lived in interphase cells as the wild-type protein, and it is still degraded in response to ionizing radiation, thus suggesting that Cdc25A may be targeted for degradation by a dual mechanism. Furthermore, Cdc25A degradation both in cycling cells and in response to DNA damage depends on phosphorylation events (Falck (2001), supra; Zhao, supra; Sorensen, supra; and Mailand, supra), a requirement for efficient target recruitment to SCF (Skp1/Cullin/F-box) ubiquitin ligases by F-box proteins (Jackson & Eldridge, Mol Cell, 2002;9:923-5; Patton et al., Trends Genet, 1998;14:236-43) as reported for other cell cycle regulators (reviewed in Spruck et al., Cell Cycle, 2002;1:250-4). Interestingly, a gain of function mutation in a C. elegans cdc25 gene has been described to result in a deregulated hyperproliferation of intestinal cells (Clucas, C., et al., EMBO J.,2002;21:665-74). The encoded mutated protein carries a Ser-to-Phe substitution within a putative DSG consensus.
However, despite these advances in the art, the F-box protein specifically responsible for targeting Cdc25A to degradation, has not been identified. Since Cdc25A is a key component in stalling the cell cycle in response to DNA damage, identification of the specific F-box protein can yield important tools in regulating Cdc25A and thereby the cell cycle, as well as the cellular response to DNA damage. This invention addresses these and other needs in the art.